One of the most exciting advances in understanding signal transduction by membrane receptors is the recent identification of the tyrosine kinase JAK2 as a receptor-associated signalling molecule for the growth hormone (GH) receptor and other members of the cytokine/hematopoeitin receptor superfamily (receptors for erythropoietin, interleukin-3, granulocyte macrophage colony stimulating factor, granulocyte colony stimulating factor, interferon-gamma, prolactin, interleukin-6, ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M). The cytokine receptor family comprises a large and rapidly expanding class of receptors for which the initial signalling events(s) had long been an enigma. Because proper functioning of the receptors that utilize JAK2 as a signalling molecule is vital for normal body function, including body growth, metabolism and immune function, it is imperative that we gain an understanding of the mechanisms governing activation and deactivation of JAK2. The goal of this proposal is to use biochemical techniques combined with immunological techniques, overexpression and site-directed mutagenesis to delineate the molecular mechanism by which one of these receptors, the receptor for GH, is stimulated by ligand binding to associate with and activate JAK2 and to determine the mechanism responsible for the transient nature of that activation. In Aim 1, the mechanism of JAK2 activation will be examined. The region(s) of GH receptor (GHR) and JAK2 required for GH-dependent GHR-JAK2 complex formation and activation of JAK2 will be identified. The role of GHR dimerization and JAK2 tyrosyl phosphorylation in the activation of JAK2 will be investigated as well as whether phosphorylation occurs by an inter- or intramolecular process. Determining whether activated JAK2 remains associated with GHR is the final goal of Aim I. Experiments described under Aim 2 are designed to determine whether dephosphorylation of tyrosyl residues in JAK2 deactivates JAK2 and if so, determine whether an SH2-containing tyrosine phosphatase is responsible for that dephosphorylation. Time permitting, regulation of the activation status of JAK2 by serine and/or threonine phosphorylation will also be examined. These studies will provide greatly needed insight into the initiating steps in GHR signal transduction. This insight should be of great assistance in deciphering similarities and differences in the interactions of JAK2 with its diverse activating receptors, and in designing therapeutic agents that target specific metabolic pathways regulated by GH and/or the other cytokines that signal through JAK kinases.